Lysotracker blue

A cocktail of fluorescent probes was prepared containing 75 nM LysoTracker Blue (ThermoFisher, Johannesburg, South Africa, L7525) and 100 nM SiR-tubulin (SpiroChrome, Stein am Rhein, Switzerland, CY-SC002) in culture media. Media was refreshed with culture media containing the dyes and the respective drugs; 10 nM Bafilomcyin (LKT labs, B0026), 200 nM Rapamycin (Sigma, R8781) and 20 nM Spermidine (Sigma, S2626). For autophagic flux analysis, saturating concentrations of Bafilomycin (400 nM) were utilized, and autophagosome pool size quantified as previously described [21 ]. Cells were treated for 8 h and then either imaged or harvested for western blot analysis.

Typically, 20,000 HeLa cells in 200 µL were seeded on µ-Slide 8 well-ibiTreat chambers (1 cm²per well, Ibidi, Germany) at least 12 h before the particle exposure. UCNPs were diluted to a concentration of 2.5 nM in cell media. After 3 h, cells were cleaned with PBS (3×) in order to remove non-associated particles. 200 µL of supplemented DMEM without phenol red were added to the cells before imaging on an Andor Dragonfly spinning disk confocal system mounted on a Nikon TiE microscope equipped with a Zyla 4.2 PLUS camera (Andor, Oxford Instruments) and an OKO-lab incubator to keep cells at 37 °C during experiment time. RB channel was obtained with an excitation of a 561 nm laser and a 620/60 nm filter. Ce6 emission was collected exciting with a 405 nm laser and using a 725/40 nm filter. All the images were processed with the free software ImageJ. For colocalization experiments lysosomes were labeled with lysotracker blue (Ex. 405, Em. 450/50, Thermofisher) and Mitotracker Green FM (Ex. 488, Em. 525/50, Thermofisher) following the manufacturer instructions.

For lysosome detection, BRGCs from 40 to 44 h L3 larvae were dissected in 1X PBS buffer. Samples were incubated for 30 min with LysoTracker Blue (Thermo Fisher Scientific, # L7525) a 1:10,000 dilution. Samples were incubated in the dark at room temperature, and on a shaker with slow agitation. Samples were then washed 3X with cold 1X PBS buffer (5 min each) and mounted in anti-Fade fluorescence mounting medium (Abcam, #ab104135). All images were taken with A Nikon C2si Confocal Microscope.

Primary antibodies for hexokinase-II, Parkin, COX-IV, VDAC, lamin A/C, Rho-GDI, α-actinin, cleaved-caspase-3, cleaved caspase-9, and LC-3B were from Cell Signaling Technology; PINK1 and BAG2 were from Novus Biologicals; GFP and ubiquitin were from Santa Cruz Biotechnology; and BAG5 was from Abcam. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), Bafilomycin A1 (BFA), 3-bromopyruvate (3BP), sodium iodoacetate, Evans blue and triphenyltetrazolium chloride (TTC) were purchased from MilliporeSigma. DharmaFECT 1 and LysoTracker Blue were purchased from Thermo Fisher Scientific.

Experiments with ATII cells were performed in bath solution (in mM: 140 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 5 glucose, 10 Hepes; pH 7.4). Cells were stimulated for secretion with 100 μM ATP (Sigma, Schnelldorf, Germany). Fusions were detected by staining secretory vesicles with Lysotracker Blue (100 nM) or LysoTracker Red (10 nM) for 20 min (Molecular Probes, Life technologies, Darmstadt, Germany). LysoTracker dyes accumulate in LBs and rapidly diffuse out of the vesicle after fusion^{52 (link)}. Microtubule polymerization was inhibited by colchicine (50 µM for 3 h) or nocodazole (60 µM for 30 min), and actin polymerization was inhibited by latrunculin B (10 µM in the experimental bath solution), all from Sigma Aldrich (Steinheim, Germany). Small GTPases were inhibited by B-toxin (300 ng/ml, for 24 h; Merck Millipore, Billerica, USA) and Rho-Inhibitor (2 μg/ml, 12 to 24 h, Cytoskeleton, Inc., Denver, USA).